Viable Pathogens Research Articles

The Application of Bacteriophage Diagnostics for Bacterial Pathogens in the Agricultural Supply Chain: From Farm-to-Fork

Bacteriophages (phages) have great potential not only as therapeutics but as diagnostics. Indeed, they have been developed and used to diagnose and detect bacterial infections, primarily in human clinical settings. The ability to rapidly detect and control bacterial pathogens in agriculture is of primary importance to maintain food security, improve animal health, and prevent the passage of zoonotic pathogens into the human population. Culture-based detection methods are often labor-intensive, and require further confirmatory tests, increasing costs and processing times needed for diagnostics. Molecular detection methods such as polymerase chain reaction are commonly used to determine the safety of food, however, a major drawback is their inability to differentiate between viable and nonviable bacterial pathogens in food. Phage diagnostics have been proven to be rapid, capable of identifying viable pathogens and do not require cultivation to detect bacteria. Phage detection takes advantage of the specificity of interaction between phage and their hosts. Furthermore, phage detection is cost effective, which is vitally important in agricultural supply chains where there is a drive to keep costs down to ensure that the cost of food does not increase. The full potential of phage detection/diagnostics is not wholly realized or commercialized. This review explores the current use and potential future scope of phage diagnostics and their application to various bacterial pathogens across agriculture and food supply chains.

The state of laboratory diagnosis, surveillance, and response to outbreaks of bacterial Meningitis, Brong Ahafo Region, Ghana, 2019

Background: One goal of WHO and Global Health Security Agenda (GHSA), is to prevent, rapidly detect, diagnose, notify and respond to infectious diseases, in order to contain them at source, and prevent spread. We assessed cerebrospinal fluid (CSF) sample management, referral, laboratory diagnostics, notification, response, and surveillance capacity in relation to bacterial meningitis in Brong Ahafo Region(BAR), Ghana. Methods and materials: We conducted a cross-sectional study in BAR in September 2019. We reviewed Meningitis case-based forms, laboratory registers, from January to August 2019, and interviewed laboratory personnel. We collected data on sample quality, type of sample container, time of collection, receipt, and outcome of culture and PCR results at the Brong Ahafo Regional Hospital Laboratory (BARHL), and the PCR Zonal Public Health Reference Laboratory (ZPHRL), in the Northern Region, respectively Results: A total of 87 CSF samples were received at BARHL, between January–August 2019, from peripheral districts in BAR. The median time for samples receipt was; 1 day;(range; 1–4). Eight, (9.2%) tested culture-positive; Streptococcus pneumoniae. Sixty-nine (79.3%) samples met the criteria for adequacy and PCR analysis at the ZPHRL. It took 21 days to receive PCR results feedback. In all, 21 (30.4%) of the 69 samples were positive by PCR; (Streptococcus pneumoniae-20, Neisseria meningitidis-1). More than half; 13(62%) of the 21 positives by PCR, had been previously declared; ‘Culture Negative’ at BARHL. Across districts, there was lack of Gram stain and rapid latex agglutination test kits. Standard sample collection tubes such as dry cryo-tubes and trans-isolate bottles (meant to sustain the viability of CSF pathogens whilst in transit) was inadequate, and often yielded contaminated results due to inappropriate handling techniques. Conclusion: Poor specimen management and weak laboratory capacity possibly accounted for the low yield of culture results at BARHL. Observation suggests a high potential of failure to rapidly diagnose, notify, respond, and contain infectious outbreaks at source. Strengthening laboratory capacity at local and district levels in Brong Ahafo Region is crucial.

Inflammatory Mediator Profiles in Secretory Otitis Media in Relationship to Viable Bacterial Pathogens and Bacterial and Viral Nucleic Acids.

Secretory otitis media (SOM) is characterized by persistence of fluid in the middle ear, often following an episode of acute otitis media. Our hypothesis is that failure to eliminate bacterial or viral pathogens may result in persistent low-grade inflammation. In this study, we analyzed inflammatory mediators in middle ear fluids from 67 children with SOM. This was combined with determinations of viable bacteria by culture along with detection of bacterial and viral genetic material by real-time polymerase chain reaction (PCR). The inflammatory mediators found at the highest concentrations (>30 ng/mL) were stem cell growth factor-β (median 110 ng/mL), CXCL1, IL-16, IL-8, migration inhibitory factor, CXCL10, and CXCL9. Among bacterial pathogens, Moraxella catarrhalis and Haemophilus influenzae dominated, regardless of detection methods, while rhinovirus dominated among viral pathogens. Middle ear fluid levels of interleukin (IL)-1α, IL-17, IL-1β, fibroblast growth factor basic, and tumor necrosis factor correlated strongly with presence of bacteria detected either by culture or PCR, while IL-1RA, IL-3, IL-6, IL-8, CCL3, CCL4, and granulocyte-colony stimulating factor correlated significantly with real-time PCR values. CXCL10, CXCL9, CCL2, and TRAIL correlated significantly with viral nucleic acid levels. To conclude, persistence of viral and bacterial pathogens may fuel persistent inflammation in SOM. Bacteria caused a broad inflammatory response, while viruses chiefly elicited the interferon-induced chemokines CXCL9 and CXCL10.

Survivability of Salmonella Pathogens and Physicochemical Characteristics of Powder Goat Milk Stored under Different Storage Treatment Regimens

Survivability of Salmonella pathogens in commercial powdered goat milk (PGM) under different storage treatments was investigated using three batches of PGM products stored at two temperatures (4 °C and 25 °C) and ten storage periods (0, 3, 7, 14, 21, 30, 60, 90, 120 and 180 days). A cocktail of three Salmonella serotypes (Salmonella agona, Salmonella enteritidis and Salmonella tennessee) was inoculated to the PGM samples and then survival of Salmonella counts was enumerated in the inoculated and non-inoculated control groups. Results showed that the initial Salmonella counts were 7.103 Log CFU (colony forming unit)/g at both temperatures. At the first 3 days, the viable Salmonella counts were reduced about 0.94 and 1.40 Log CFU/g at 4 °C and 25 °C, respectively, where the same levels were sustained for 14 days. Further reductions continued and at the end of 180 days storage, Salmonella survivability was 1.15 Log CFU/g higher at 4 °C than at 25 °C under the same water activity condition. As the storage period advanced, viable pathogen counts were gradually decreased. The pH of samples stored at 4 °C for 0 and 4 month were higher than those stored at 25 °C except for 2 months, while no differences were found in water activity (aw) between treatments of the PGM products. With regard to physicochemical characteristics, the samples stored at 25 °C showed higher POV (peroxide value) values than those stored at 4 °C for 2 and 4 month periods, indicating that the rate of lipid oxidation in the PGM was elevated by a higher storage temperature and a longer storage period. The basic nutrient compositions of the experimental PGM were similar to those reported in recent studies. Oleic acid (C18:1) was the highest, caprylic acid (C8:0) was the second highest, and behenic acid (C22:0) was the lowest concentration among all fatty acids identified in the PGM samples. Most of the fatty acid concentrations tended to decrease with advanced storage periods. This research indicates that the survivability of Salmonella pathogens in the PGM products stored at 4 °C for 180 days was higher than those stored at 25 °C under the same aw condition.

Mammalian Cell-Based Immunoassay for Detection of Viable Bacterial Pathogens.

Rapid detection of live pathogens is of paramount importance to ensure food safety. At present, nucleic acid-based polymerase chain reaction and antibody-based lateral flow assays are the primary methods of choice for rapid detection, but these are prone to interference from inhibitors, and resident microbes. Moreover, the positive results may neither assure virulence potential nor viability of the analyte. In contrast, the mammalian cell-based assay detects pathogen interaction with the host cells and is responsive to only live pathogens, but the short shelf-life of the mammalian cells is the major impediment for its widespread application. An innovative approach to prolong the shelf-life of mammalian cells by using formalin was undertaken. Formalin (4% formaldehyde)-fixed human ileocecal adenocarcinoma cell line, HCT-8 on 24-well tissue culture plates was used for the capture of viable pathogens while an antibody was used for specific detection. The specificity of the Mammalian Cell-based ImmunoAssay (MaCIA) was validated with Salmonella enterica serovar Enteritidis and Typhimurium as model pathogens and further confirmed against a panel of 15 S. Enteritidis strains, 8 S. Typhimurium, 11 other Salmonella serovars, and 14 non-Salmonella spp. The total detection time (sample-to-result) of MaCIA with artificially inoculated ground chicken, eggs, milk, and cake mix at 1–10 CFU/25 g was 16–21 h using a traditional enrichment set up but the detection time was shortened to 10–12 h using direct on-cell (MaCIA) enrichment. Formalin-fixed stable cell monolayers in MaCIA provide longer shelf-life (at least 14 weeks) for possible point-of-need deployment and multi-sample testing on a single plate.

Environmental DNA from multiple pathogens is elevated near active Atlantic salmon farms

The spread of infection from reservoir host populations is a key mechanism for disease emergence and extinction risk and is a management concern for salmon aquaculture and fisheries. Using a quantitative environmental DNA methodology, we assessed pathogen environmental DNA in relation to salmon farms in coastal British Columbia, Canada, by testing for 39 species of salmon pathogens (viral, bacterial, and eukaryotic) in 134 marine environmental samples at 58 salmon farm sites (both active and inactive) over 3 years. Environmental DNA from 22 pathogen species was detected 496 times and species varied in their occurrence among years and sites, likely reflecting variation in environmental factors, other native host species, and strength of association with domesticated Atlantic salmon. Overall, we found that the probability of detecting pathogen environmental DNA (eDNA) was 2.72 (95% CI: 1.48, 5.02) times higher at active versus inactive salmon farm sites and 1.76 (95% CI: 1.28, 2.42) times higher per standard deviation increase in domesticated Atlantic salmon eDNA concentration at a site. If the distribution of pathogen eDNA accurately reflects the distribution of viable pathogens, our findings suggest that salmon farms serve as a potential reservoir for a number of infectious agents; thereby elevating the risk of exposure for wild salmon and other fish species that share the marine environment.

Detection and distribution of vbnc/viable pathogenic bacteria in full-scale drinking water treatment plants

Viable but non-culturable (VBNC) bacteria have attracted widespread attention since they are inherently undetected by traditional culture-dependent methods. Importantly, VBNC bacteria could resuscitate under favorable conditions leading to significant public health concerns. Although the total number of viable bacteria has been theorized to be far greater than those that can be cultured, there have been no reports quantifying VBNC pathogenic bacteria in full-scale drinking water treatment plants (DWTPs). In this work, we used both culture-dependent and quantitative PCR combination with propidium monoazide (PMA) dye approaches to characterize cellular viability. Further, we established a method to quantify viable pathogens by relating specific gene copies to viable cell numbers. Ratios of culturable bacteria to viable 16S rRNA gene copies in water and biological activated carbon (BAC) biofilms were 0–4.75% and 0.04–56.24%, respectively. The VBNC E. coli, E. faecalis, P. aeruginosa, Salmonella sp., and Shigella sp. were detected at levels of 0–103 cells/100 mL in source water, 0–102 cells/100 mL in chlorinated water, and 0–103 cells/g in BAC biofilms. In addition, differences between the total and viable community structures after ozonation and chlorination were investigated. The relative abundance of opportunistic pathogens such as Mycobacterium, Sphingomonas, etc. increased in final water, likely due to their chlorine resistance. In summary, we detected significant quantities of viable/VBNC opportunistic pathogens in full-scale DWTPs, confirming that traditional, culture-dependent methods are inadequate for detecting VBNC bacteria. These findings suggest a need to develop and implement rapid, accurate methods for the detection of VBNC pathogenic bacteria in DWTPs to ensure the safety of drinking water.

The use of non‐surgical interventions in patients with peri‐implantitis; a systematic review and meta‐analysis

AbstractObjectivesTo systematically assess the efficacy of different non‐surgical treatment methods to manage peri‐implantitis reported to date in the literature, together with its correlation with time following therapy.Materials and MethodsA systematic literature review was undertaken to identify randomised control trials of the non‐surgical management of peri‐implantitis published up to November 2019. The search was limited to English language human studies containing follow‐up periods of ≥3 months and for sample size of 10 or more patients. A meta‐analysis was implemented for the following clinical parameters: Peri‐implant pocket depth (PPD), bleeding on probing (BOP), clinical attachment level (CAL), radiographic bone loss (RBL) and mucosal recession (MR).ResultsTwelve articles met the inclusion criteria. Two principal treatment modalities were identified; mechanical debridement and laser therapy, with two adjunctive therapies antimicrobial and antiseptic agents. Non‐surgical interventions (ultrasonic scalers, Er:YAG laser and powdered air‐abrasive devices) showed significant clinical improvement in the short term (<3 months). Clinical benefit was demonstrated with the adjunctive use of antimicrobial agents in the short term but diminished with time. Antiseptic agents alone have no significant effect. Non‐surgical therapies applied in these studies failed to arrest mucosal recession, peri‐implant bone loss or reduce the counts of viable pathogens in the long term.ConclusionThe evidence demonstrate that the clinical parameters of peri‐implantitis, i.e. BOP, PPD and CAL may all be improved by simple mechanical debridement, using either ultrasonic instrumentation or Er:YAG laser therapy; adjunctive antimicrobial and antiseptic therapy. Further randomised control trials in this area are, however, required.

Ефективність використання дигестату біогазових установок

It has been established that the digestate of biogas plants is a valuable organic fertilizer that is quickly absorbed by plants, contains a complex of macro- and microelements necessary for plant growth, has a high content of readily available nitrogen for plants, has a pH level close to neutral and does not contain viable weed seeds and pathogens. microflora. It is technically difficult to make untreated digestate into the soil, so it is predivided into solid and liquid fractions on separators. A major problem with the commercial use of digestate in Ukraine is the lack of its standardization. When applying digestate to the company's own fields, which owns the biogas plant, the standardization of its quality is not critical. However, the lack of a standard for digestate as a high-quality organic fertilizer significantly limits its use. The needs for digestate were determined to restore soil fertility and fertilize crops in and with a herd of 1,000 cattle (500 heads of dairy cows and 500 heads of young cattle and heifers) and with an arable land area of 1,800 hectares. It is established that only one ton of digestate remains for commercial use after application to the fields, the profit from the sale of which does not significantly affect the profitability of biogas production. The payback period of the biogas plant is 6.4 years and depends on the cost of biogas production. With the commercial use of all digestate produced during the year, the payback period of the biogas plant is reduced to one year and below and does not depend on the cost of biogas production, and the impact of the "green" tariff on the payback period of the biogas plant is leveled.

An Optimized Method to Assess Viable Escherichia coli O157:H7 in Agricultural Soil Using Combined Propidium Monoazide Staining and Quantitative PCR.

Agricultural soil contaminated by manure is becoming an important source for the transmission of foodborne pathogens. There is an urgent need for a rapid and accurate method for viable pathogen detection in agricultural soil samples. Propidium monoazide (PMA) is a DNA-binding dye that can inhibit the amplification of DNA from dead cells through subsequent quantitative polymerase chain reaction (qPCR), thus allowing for viable cells detection and quantification. The objective of this study was to detect viable Escherichia coli O157:H7 in the agricultural soils by PMA-qPCR. In this study, cell extraction and gradient density centrifugation were incorporated before PMA-qPCR to reduce the interference of soil particle including turbidity and a high ratio of dead cells. The optimized treatment conditions were determined as follows, the maximum removal of DNA from dead cells was achieved by 1.067 g/mL Percoll of centrifugation and 50 μM PMA treatment. Under these conditions, the turbidity of paddy soil suspensions decreased from 3500 to 28.4 nephelometric turbidity units (NTU), and the ratio of viable cells to dead cells increased from 0.001 to 1.025%. For typical agricultural soils collected in China, as low as 102colony-forming units (CFU)/g of viable cells could be accurately detected in the presence of a large number of dead cells (107 CFU/g) by the optimized PMA-qPCR. Significantly, with comparable accuracy, the optimized PMA-qPCR assay was more sensitive, accessible and rapid than conventional culture methods. In addition, the viable but non-culturable (VBNC) state of E. coli O157:H7 cells in paddy soils, which often escaped the detection by conventional culture methods, could be quantitatively characterized by the optimized PMA-qPCR method. Potentially, the optimized PMA-qPCR can be further applied for viable pathogens detection and give insight into the prevalence of VBNC E. coli O157:H7 in agricultural soil.

The risk of infectious pathogens in breast-feeding, donated human milk and breast milk substitutes.

This review collates the published reports that focus on microbial and viral illnesses that can be transmitted by breast milk, donor milk and powdered infant formula (PIF). In this context, we attempt to define a risk framework encompassing those hazards, exposure scenarios, vulnerability and protective factors. A literature search was performed for reported cases of morbidity and mortality associated with different infant feeding modes. Exclusive breast-feeding is the recommended for infant feeding under 6 months, or failing that, provision of donated human milk. However, the use of PIF remains high despite its intrinsic and extrinsic risk of microbial contamination, as well as the potential for adverse physiological effects, including infant gut dysbiosis. Viable pathogen transmission via breast-feeding or donor milk (pasteurised and unpasteurised) is rare. However, transmission of HIV and human T-cell lymphotropic virus-1 is a concern for breast-feeding mothers, particularly for mothers undertaking a mixed feeding mode (PIF and breast-feeding). In PIF, intrinsic and extrinsic microbial contamination, such as Cronobacter and Salmonella, remain significant identifiable causes of infant morbidity and mortality. Disease transmission through breast-feeding or donor human milk is rare, most likely owing to its complex intrinsically protective composition of human milk and protection of the infant gut lining. Contamination of PIF and the morbidity associated with this is likely underappreciated in terms of community risk. A better system of safe donor milk sharing that also establishes security of supply for non-hospitalised healthy infants in need of breast milk would reduce the reliance on PIF.

Resuscitation of the Helicobacter pylori Coccoid Forms by Resuscitation Promoter Factor Obtained from Micrococcus Luteus.

Helicobacter pylori is a gram negative, spiral-shaped, and microaerophilic bacteria which can cause life-threatening diseases. It is known that more than 55% of the human population in the world is already infected by this bacterium. The traditional treatment of H. pylori infection consists of a combination of two or more antibiotics. However, H. pylori has evolved to turning its shape from spiral to coccoid form in the presence of antibiotics and this decreases the therapeutic efficacies of conventional antibiotic applications. Resuscitation promoter factor (RPF) is a protein secreted by Micrococcus luteus have significant resuscitation effects on some bacteria especially in the group of viable but non-culturable (VBNC) pathogens. However, there is no study in the literature investigating the resuscitation effects of RPF derived from M. luteus on H. pylori in order to change its form from coccoid to spiral. The purpose of this study is to investigate the resuscitation effect of RPF-containing metabolites isolated from M. luteus on the morphological transformation of H. pylori coccoid forms to spiral forms in order to increase their susceptibilities to antibiotic treatments. Rpf-containing metabolites were primarily obtained from M. luteus culture supernatants. H. pylori was exposed to five different conditions such as prolonged culture, incubation at + 4 °C, incubation at + 22°C, cultivation in PBS and treatment with kanamycin in order to induce transformations of bacteria to coccoid forms. Induced H.pylori coccoids were characterized by inverted microscope, UV spectrophotometer, SEM imaging, and flow-cytometer. As a result, it was found that the most suitable condition for inducing coccoid forms was cultivation of bacteria with kanamycin. Followingly, different concentrations of RPF-containing metabolites were applied on H. pylori coccoids induced by kanamycin. For the first time in this study, it was determined that the Rpf-containing metabolites obtained from M. luteus demonstrated very high resuscitation effect on kanamycin-induced H. pylori coccoid forms. This new approach for resuscitation of H. pylori coccoids is thought to play an important role in increasing the treatment effectiveness of the conventional antibiotics against the infection.

Immune-modulatory Properties of the Octapeptide NAP in Campylobacter jejuni Infected Mice Suffering from Acute Enterocolitis.

Human infections with the food-borne zoonotic pathogen Campylobacter jejuni are progressively rising and constitute serious global public health and socioeconomic burdens. Hence, application of compounds with disease-alleviating properties are required to combat campylobacteriosis and post-infectious sequelae. In our preclinical intervention study applying an acute C. jejuni induced enterocolitis model, we surveyed the anti-pathogenic and immune-modulatory effects of the octapeptide NAP which is well-known for its neuroprotective and anti-inflammatory properties. Therefore, secondary abiotic IL-10−/− mice were perorally infected with C. jejuni and intraperitoneally treated with synthetic NAP from day 2 until day 5 post-infection. NAP-treatment did not affect gastrointestinal C. jejuni colonization but could alleviate clinical signs of infection that was accompanied by less pronounced apoptosis of colonic epithelial cells and enhancement of cell regenerative measures on day 6 post-infection. Moreover, NAP-treatment resulted in less distinct innate and adaptive pro-inflammatory immune responses that were not restricted to the intestinal tract but could also be observed in extra-intestinal and even systemic compartments. NAP-treatment further resulted in less frequent translocation of viable pathogens from the intestinal tract to extra-intestinal including systemic tissue sites. For the first time, we here provide evidence that NAP application constitutes a promising option to combat acute campylobacteriosis.

Poultry litter as potential source of pathogens and other contaminants in groundwater and surface water proximal to large-scale confined poultry feeding operations

Manure from livestock production has been associated with the contamination of water resources. To date, research has primarily focused on runoff of these contaminants from animal operations into surface water, and the introduction of poultry-derived pathogenic zoonoses and other contaminants into groundwater is under-investigated. We characterized pathogens and other microbial and chemical contaminants in poultry litter, groundwater, and surface water near confined poultry feeding operations (chicken layer, turkey) at 9 locations in Iowa and one in Wisconsin from May and June 2016. Results indicate that poultry litter from large-scale poultry confined feeding operations is a likely source of environmental contamination and that groundwater is also susceptible to such poultry-derived contamination. Poultry litter, groundwater, and surface water samples had detections of viable bacteria growth (Salmonella spp., enterococci, staphylococci, lactobacilli), multi-drug resistant Salmonella DT104 flost and int genes, F+ RNA coliphage (group I and IV), antibiotic resistance genes (ARGs; blaDHA, blaOXA-48, blaTEM, blaCMY-2, tetM), phytoestrogens (biochanin A, daidzein, formononetin), and a progestin (progesterone). In addition, mcr-1 (a colistin ARG), was detected in a groundwater sample and in another groundwater sample, antibiotic resistant isolates were positive for Brevibacterium spp., a potential signature of poultry in the environment. Detectable estrogenicity was not measured in poultry litter, but was observed in 67% of the surface water samples and 22% were above the U.S. Environmental Protection Agency trigger level of 1 ng/L. The transport of microbial pathogens to groundwater was significantly greater (p < 0.001) than the transport of trace organic contaminants to groundwater in this study. In addition to viable pathogens, several clinically important ARGs were detected in litter, groundwater, and surface water, highlighting the need for additional research on sources of these contaminants in livestock dominated areas.

Fate of Listeria monocytogenes in the Presence of Resident Cheese Microbiota on Common Packaging Materials.

Literature data regarding the survival of microorganisms on materials used for food package purposes are scarce. The aim of the current study is to assess the survival of Listeria monocytogenes on different packaging materials for dairy products during extended storage at different temperatures. Three packaging materials (5 × 5 cm) were contaminated with a cocktail of five strains of Listeria monocytogenes suspended in a cheese homogenate, including the cheese’s native microbial population. Contaminated samples were incubated at 37°, 12°, and 4°C and periodically analyzed up to 56 days. The evolution of the total viable count and pathogen population was evaluated. At 37°C, the results showed that Listeria monocytogenes was no longer detected on polyethylene-coated nylon (B) by day 4 and on polyethylene-coated parchment (A) and greaseproof paper (C) by day 7. Interestingly, the initial cell population (ranging between 2.5 and 2.7 log CFU/cm2) of Listeria monocytogenes increased to 3 log CFU/cm2 within 4 days of storage at 12°C on A and C. During storage, the number remained fairly constant at 12°C and 4°C on two materials (A–C) and decreased slowly on the third one (B). This study shows that survival of Listeria monocytogenes on packaging materials for dairy products will be higher when stored at 4 or 12°C compared to 37°C. The survival of Listeria monocytogenes on the packaging materials raises concerns of cross-contamination during food handling and preparation at catering and retail premises and within the home, highlighting the importance of treating the packaging materials as a potential source of cross-contamination. These initial findings may aid in quantifying risks associated with contamination of food packaging materials.

Direct Metatranscriptome RNA-seq and Multiplex RT-PCR Amplicon Sequencing on Nanopore MinION - Promising Strategies for Multiplex Identification of Viable Pathogens in Food.

Viable pathogenic bacteria are major biohazards that pose a significant threat to food safety. Despite the recent developments in detection platforms, multiplex identification of viable pathogens in food remains a major challenge. A novel strategy is developed through direct metatranscriptome RNA-seq and multiplex RT-PCR amplicon sequencing on Nanopore MinION to achieve real-time multiplex identification of viable pathogens in food. Specifically, this study reports an optimized universal Nanopore sample extraction and library preparation protocol applicable to both Gram-positive and Gram-negative pathogenic bacteria, demonstrated using a cocktail culture of E. coli O157:H7, Salmonella enteritidis, and Listeria monocytogenes, which were selected based on their impact on economic loss or prevalence in recent outbreaks. Further evaluation and validation confirmed the accuracy of direct metatranscriptome RNA-seq and multiplex RT-PCR amplicon sequencing using Sanger sequencing and selective media. The study also included a comparison of different bioinformatic pipelines for metatranscriptomic and amplicon genomic analysis. MEGAN without rRNA mapping showed the highest accuracy of multiplex identification using the metatranscriptomic data. EPI2ME also demonstrated high accuracy using multiplex RT-PCR amplicon sequencing. In addition, a systemic comparison was drawn between Nanopore sequencing of the direct metatranscriptome RNA-seq and RT-PCR amplicons. Both methods are comparable in accuracy and time. Nanopore sequencing of RT-PCR amplicons has higher sensitivity, but Nanopore metatranscriptome sequencing excels in read length and dealing with complex microbiome and non-bacterial transcriptome backgrounds.

Species-specific secretion of ESX-5 type VII substrates is determined by the linker 2 of EccC5.

Mycobacteria use type VII secretion systems (T7SSs) to translocate a wide range of proteins across their diderm cell envelope. These systems, also called ESX systems, are crucial for the viability and/or virulence of mycobacterial pathogens, including Mycobacterium tuberculosis and the fish pathogen Mycobacterium marinum. We have previously shown that the M. tuberculosis ESX‐5 system is unable to fully complement secretion in an M. marinum esx‐5 mutant, suggesting species specificity in secretion. In this study, we elaborated on this observation and established that the membrane ATPase EccC5, possessing four (putative) nucleotide‐binding domains (NBDs), is responsible for this. By creating M. marinum‐M. tuberculosis EccC5 chimeras, we observed both in M. marinum and in M. tuberculosis that secretion specificity of PE_PGRS proteins depends on the presence of the cognate linker 2 domain of EccC5. This region connects NBD1 and NBD2 of EccC5 and is responsible for keeping NBD1 in an inhibited state. Notably, the ESX‐5 substrate EsxN, predicted to bind to NBD3 on EccC5, showed a distinct secretion profile. These results indicate that linker 2 is involved in species‐specific substrate recognition and might therefore be an additional substrate recognition site of EccC5.

Bacteriostatic Effect of Multidose Preservative-free Buffered Saline Used in Scleral Lens Wear.

Scleral lenses have become an increasingly common treatment for ocular surface disease and irregular corneas. Multidose, preservative-free saline solutions are frequently used off-label to fill scleral lenses. Because the fluid resides over the ocular surface during lens wear, contaminated solutions may increase the risk of infectious complications. We sought to assess the viability of skin microorganisms and pathogens associated with keratitis once introduced into a multidose preservative-free saline (MDPFS) solution containing the bacteriostatic agent boric acid (PuriLens Plus; The Lifestyle Co., Inc., Freehold, NJ). Eleven bacterial and one yeast isolate were each inoculated to three lots of MDPFS as well as to sterile normal saline for comparison. Microorganism concentrations were enumerated at baseline and days 1, 3, 7, 14, 21, and 28. Persistence of microorganism viability was compared between MDPFS lots and between MDPFS and normal saline for each organism. Duration of microorganism viability was ≥24 hours in MDPFS with no significant difference in the distribution of survival duration of microorganisms in MDPFS versus normal saline (P = .15). Candida albicans concentrations declined 14 days earlier in MDPFS, whereas concentrations of viable organisms in MDPFS remained within 1 log of baseline for the longest durations for Pseudomonas aeruginosa (7 days), Escherichia coli (14 days), and Achromobacter xylosoxidans (≥28 days). Gram-positive organism concentrations remained within 1 log of baseline for no more than 3 days. Mild lot-to-lot variation in organism concentrations was noted near the end points of viability. Bacteriostasis was demonstrated in that concentrations of all organisms remained at or below baseline levels throughout the 28-day period. After microbial contamination, persistence of organism viability was similar in PuriLens and normal saline. Environmental gram-negative organisms, many of which can contribute to infectious keratitis, can persist for weeks once introduced into saline solutions.

Investigations into Enhancing Yersinia pestis Cells Viability following Environmental Sampling for Forensic Analysis.

Following an intentional or accidental bio-warfare agent (BWA) release, environmental sample analysis is absolutely critical to determine the extent of contamination. When dealing with nonspore forming BWA (e.g., Yersinia pestis), retention of cell viability is central to such analyses. Even though significant advances have been achieved in DNA sequencing technologies, a positive identification of BWAs in environmental samples must be made through the ability of cells to form colony-forming units upon culturing. Inability to revive the cells between collection and analysis renders such studies inconclusive. Commercial kits designed to preserve the viability of pathogens contained within clinical samples are available, but many of them have not been examined for their ability to preserve samples containing suspected BWAs. The study was initiated to examine the applicability of commercial solutions aiding in retention of Y.pestis viability in samples stored under nonpermissive temperatures, that is, 40 and 37°C. While none of the tested solutions sustained cell viability at 40°C, the results show five out of 17 tested preservatives were capable of supporting viability of Y.pestis at 37°C.

Вплив абіотичних факторів на життєздатність і розвиток екзогенних стадій – ооцист еймерій птахів

Modern high-tech and sanitary-hygienic support for the poultry industry is impossible without therapeutic and preventive measures against eimeriosis. Today, the focus is on the fight against the endogenous stages of the development of eimeria in the body of birds with the help of a huge arsenal of chemicals or immunoprophylaxis. To destroy the exogenous stages of the development of eimeria - oocysts in the external environment the number of disinvasing substances is quite small. The effect of abiotic factors on the development and viability of pathogens - Aimeria oocysts has not been fully studied. Objective. To determine the influence of abiotic factors (temperature, humidity) on the viability and development of oocysts of Aimeria birds. The studies were carried out in the laboratory of the Department of Parasitology of Kharkiv State Zooveterinary Academy in vitro, using conventional flotation and microscopic research methods. Material for the study was selected from patients experimentally invaded by chicken eimeriosis during defecation. Feces were studied by the Fulleborn flotation method, feces samples were placed in Petri dishes, kept in a thermostat at +18, + 20 ° С. The drying process (moisture loss) was monitored by weighing, and the development and viability of Eimeria oocysts were microscopical. The counting of the number of non-sporulated and sporulated oocysts in 1 g of feces using a Goryaev camera was carried out. As a result of the study, it was found that the drying of feces negatively affects the development and viability of bird eimeria oocysts. With a decrease in moisture of 31.97 ± 1.24% and 41.67 ± 0.47% in feces, respectively, from 65.8 ± 0.7 to 80.6 ± 0.9% oocysts die, and with moisture loss of 65, 48 ± 1.35% and 70.9 ± 1.2% and from 91.4 ± 0.7% to 100% Aimeria oocysts die.